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"NEURODEGENERATION AND REGENERATION IN RODENT MODELS OF PEDIATRIC TRAUMATIC BRAIN INJURY"

by
Matthew Goodus
Integrative Neurosciences Program
B.S. 2002, University of California, Irvine


Thesis Advisor: Steven W. Levison, Ph.D.
Professor
Department of Neurology & Neurosciences

Friday, January 9, 2015
9:30 A.M., Cancer Center, G-1196


Abstract

Pediatric traumatic brain injury is a significant problem that affects many children each year. Progress is being made in developing neuroprotective strategies to combat these injuries; however, investigators are a long way from therapies to fully preserve injured neurons and glia. Here we used two different models of pediatric brain injury to gain new insights into regenerative and degenerative processes set into motion by traumatic injury to the CNS.
Given the importance of stem cells in repairing damaged tissues, and the known persistence of neural precursors in the subventricular zone (SVZ) we evaluated regenerative responses of the SVZ to a focal brain lesion. As tissues repair more slowly with aging, injury responses of male Sprague Dawley rats at 6,11,17 and 60 days of age and C57Bl/6 mice at 14 days of age were compared. In injured immature animals, cell proliferation in the dorsolateral SVZ more than doubled by 48 hours. By contrast, the proliferative response was almost undetectable in the adult brain. Three approaches were used to assess the relative numbers of bona fide neural stem cells; the neurosphere assay (on rats injured at P11), flow cytometry using a novel 4-marker panel (on mice injured at P14) and staining for stem/progenitor cell markers in the niche (on rats injured at P17). Precursors from the injured immature SVZ formed almost twice as many spheres as precursors from un-injured age matched brains. Furthermore, spheres formed from the injured brain were larger indicating that the neural precursors that formed these spheres divided more rapidly. Flow cytometry revealed a two-fold increase in the percentage of stem cells, a 4-fold increase in multipotential progenitor 3 cells and a 2.5-fold increase in glial restricted progenitor-2/multipotential-3 cells. Analogously, there was a two fold increase in the mitotic index of Nestin+/Mash1- immunoreactive cells within the immediately subependymal region. As the early postnatal SVZ is predominantly generating glial cells, an expansion of precursors might not necessarily lead to the production of many new neurons. On the contrary, many BrdU+/Doublecortin+ cells were observed streaming out of the SVZ into the neocortex 2 weeks after injuries to postnatal day 11 rats. However, very few new mature neurons were seen adjacent to the lesion 28 days after injury. Altogether, these data indicate that immature SVZ cells mount a more robust proliferative response to a focal brain injury than adult cells, which includes an expansion of stem cells, primitive progenitors and neuroblasts. Nonetheless, this regenerative response does not result in significant neuronal replacement indicating that new strategies need to be implemented to retain the regenerated neurons and glia that are being produced.
When the nervous system sustains injury, the daily activities of glial cells are reprogramed by the actions of extracellular signals that are released in response to damage. Leukemia inhibitory factor (LIF) is rapidly induced after CNS and PNS injuries whereupon it stimulates neurons, macroglia and microglia. Whether LIF signaling is, on balance, beneficial or detrimental for functional recovery has not been established. Here we compared the extent of neocortical and subcortical white matter damage sustained by adolescent LIF haplodeficient mice vs. wild type mice after a closed head injury. Closed head injuries were produced using a 5 mm rounded metal piston that was pneumatically delivered to the saggital suture of postnatal day l8 CD1 mice at a velocity of 5 m/s, with a depth of 1.0 mm past zero point on the skull surface with a dwell time of 150 ms, which produced a reproducible, but relatively mild injury. At 2 days of recovery, both astrogliosis and microgliosis were comparatively diminished in the LIF haplodeficient mice; however both astrogliosis and microgliosis were exacerbated at 7 and 14 days of recovery. This desynchronization of the gliotic response was accompanied by increased white and gray matter apoptosis, neuronal cell death,, reduced interhemispheric compound action potentials and hypomyelination. LIF haplodeficient mice also sustained greater callosal axonal loss and displayed more severe motor and sensory deficits at both 7 and 14 days of recovery compared to wild type mice. Altogether, these data demonstrate that LIF is an essential timing signal for both astrogliosis and microgliosis after brain injury, and that a 50% reduction in LIF expression is sufficient to elicit a cascade of events that result in a second wave neurodegeneration and more severe neurological deficits.


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